Synthesis and biological evaluation of novel luteolin derivatives as antibacterial agents

Article abstract of DOI:10.1016/j.ejmech.2008.01.013

A series of luteolin derivatives 2-20 were prepared, 3-20 of which were first reported. The chemical structures of these compounds were confirmed by means of ¹H NMR, ESI-MS and elemental analyses. The compounds were assayed for antibacterial (B

Full text of DOI:10.1016/j.ejmech.2008.01.013

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European Journal of Medicinal Chemistry 44 (2009) 908e914
http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and biological evaluation of novel luteolin
derivatives as antibacterial agents
*
Peng-Cheng Lv, Huan-Qiu Li, Jia-Yu Xue, Lei Shi, Hai-Liang Zhu
Institute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University,
Nanjing 210093, People’s Republic of China
Received 21 July 2007; received in revised form 13 December 2007; accepted 10 January 2008
Available online 25 January 2008
Abstract
A series of luteolin derivatives 2e20 were prepared, 3e20 of which were first reported. The chemical structures of these compounds were
1
confirmed by means of H NMR, ESI-MS and elemental analyses. The compounds were assayed for antibacterial (Bacillus subtilis, Staphylo-
coccus aureus, Pseudomonas fluorescens and Escherichia coli) activities by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl trtrazolium
bromide) method. Among the compounds tested, most of them displayed significant activity against the tested strains, and 2-(2,3-dihydroben-
zo[b][1,4]dioxin-6-yl)-5-hydroxy-7-(2-(3-morpholinopropylamino)ethoxy)-4H-chromen-4-one (17) showed the most favorable antibacterial
activity in vitro with MICs of 1.562, 3.125, 3.125, and 6.25 mg/mL against B. subtilis, S. aureus, P. fluorescens and E. coli, respectively.
Structureeactivity relationships (SAR) were also discussed based on the obtained experimental data.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Synthesis; Biological evaluation; Luteolin derivatives; Antibacterial agents
1. Introduction
derivatives. Based on our recent efforts on the improvement
of isoflavonoids [10e12], our research interest now is focused
on the modification of luteolin.
Luteolin
(2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-
chromen-4-one), a polyphenolic compound available in foods
of plant origin, belongs to the flavone subclass of flavonoids,
usually occurring as glycosylated forms in celery, green pep-
per, perilla leaf and camomile tea [1e3]. It has been
reported to have many different biological activities such as
antimutagenic, antiplatelet aggregation [4], antitumorigenic,
antioxidant and anti-inflammatory properties [1]. Luteolin
can also display anticancer effect [5], and inhibit a series of
human cancer cell lines (renal A-549, ovary SK-OV-3, mela-
noma SK-MEL-2, XF-498, HCT15, gastric HGC-27) [6]. In
addition, luteolin has been shown to have antibacterial activity
against a number of bacteria [7e9], however, few reports have
been dedicated to the improvement of the antibacterial activi-
ties and the structureeactivity relationships of luteolin
It is reported that the 2,3-dihydro-1,4-benzodioxin nucleus
bearing an acidic acetic moiety conferred anti-inflammatory
activity as non-steroidal acid anti-inflammatory drugs
(NSAIDS) [13]. Furthermore, it is also found that ethyl 1,4-
benzodioxan-2-carboxylate is used as an intermediate com-
pound for the production of drug doxazosin mesylate, which
is a drug for the treatment of hypertension and benign prostatic
hyperplasia [14]. It belongs to the quinazoline group of drugs
and is presently produced in the racemic form [15]. In addi-
tion, strobilurin E, which has a similar 1,4-benzodioxan struc-
ture, has been reported to have strong antifungal activity [16].
In view of realizing these results and the special structure of
luteolin which has two adjacent phenolic hydroxyl in the B
ring, we have synthesized a series of luteolin derivatives which
have the potential pharmacophore 1,4-benzodioxin and 2-
carbon spacer at C-7 position. At the same time, these deriva-
tives of luteolin were assayed for antibacterial (Bacillus
subtilis, Staphylococcus aureus, Pseudomonas fluorescens
* Corresponding author. Tel./fax: þ86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.013

P.-C. Lv et al. / European Journal of Medicinal Chemistry 44 (2009) 908e914
909
and Escherichia coli) activities by MTT method. Fortunately,
it was found that some compounds displayed high antibacte-
rial activities against the tested strains in vitro.
ring, but their antibacterial activity was different. Compound
17 displayed significant activity while 15 exhibited moderate
activity compared to the former. A possible explanation
for this result is the former had better lipophilicity than the
latter.
2. Chemistry
Among all the synthetic compounds, two species can be di-
vided, one containing heterocyclic moieties, including com-
pounds 10, 11 and 14e20, and compounds 4e9 containing
aliphatic chain substituents. From Table 1 we can see that
compounds 10, 11 and 14e20 showed obvious antibacterial
activity against the tested strains, while 4e9 show moderate
antibacterial activity. So the species containing heterocyclic
substituents have a much higher inhibitory potency than the
other species.
All the compounds prepared contain a 2-carbon spacer at
C-7 position. This modification is likely to make the com-
pounds more lipophilic, which may increase their permeability
of the cell membrane. The biological activity of a particular
substance depends on a complex sum of individual properties
including compound structure, affinity for the target site, and
survival in the medium of application, survival within the
biological system, transport properties, and state of the target
organism [17]. In this study, we focused our attention on the
structureeactivity relationships. Structural analysis of these
compounds may provide some explanation for the struc-
tureeactivity relationships.
In this paper a series of luteolin derivatives containing a 2-
carbon spacer at C-7 position are prepared, which also have
potential pharmacophore 1,4-benzodioxin. The synthesis of
compounds 2e20 followed the general pathway illustrated in
Scheme 1. Compounds 2 and 3 were the key intermediates
for synthesis of the compounds investigated. Treating com-
pound 1 with 1,2-dibromoethane at 70 ꢀC for 30 min in anhy-
drous DMF catalyzed by potassium carbonate yielded
compound 2, which has the potential pharmacophore 1,4-ben-
zodioxin. Secondly, in order to increase the antibacterial prop-
erties of luteolin, whose ring A system linked to the
alkylamine by a 2-carbon spacer at C-7 position was investi-
gated, with a view to modify their lipophilicity. Thus, deriva-
tive 3 was prepared through ‘step ii’ by treating compound 2
with excessive amounts of 1,2-dibromoethane at 120 ꢀC for
2 h in anhydrous DMF catalyzed by potassium carbonate.
Thirdly, reaction of 3 with different alkylamines and aromatic
amines in anhydrous DMF at 80 ꢀC for 1 h yielded compounds
4e20. All of the synthetic compounds gave satisfactory ana-
lytical and spectroscopic data, which were in full accordance
with their depicted structures.
4. Conclusions
3. Results and discussion
In this paper a series of luteolin derivatives containing a 2-
carbon spacer at C-7 position are prepared and evaluated for
antibacterial activity against B. subtilis, S. aureus, P. fluores-
cens and E. coli. Bioassays indicated that some of the com-
pounds showed high antibacterial activity in vitro, especially
morpholine derivative 17 was found to be more active than
others at an MIC value of 1.562, 3.125, 3.125, and 6.25 mg/
mL against B. subtilis, S. aureus, P. fluorescens and E. coli,
respectively. The study on structureeactivity relationships of
these luteolin derivatives indicated that the hydrophilicity
and aromaticity seemed to be important for the antibacterial
activity.
All the synthesized compounds were screened for antibac-
terial activity against two Gram-positive bacterial strains (B.
subtilis and S. aureus) and two Gram-negative bacterial strains
(E. coli and P. fluorescens) by MTT method. The MICs (min-
imum inhibitory concentrations) of the compounds against
four bacteria are presented in Table 1.
Compounds 13, 16 and 17 displayed prominent activity
equal to that of penicillin G against B. subtilis (1.562 mg/
mL) while compounds 10e12, 14, 15 and 18e20 exhibited
moderate activity (3.125e6.25 mg/mL). Compounds 13, 17
showed significant activity against S. aureus (3.125 mg/mL)
while compounds 12, 15 and 16 displayed moderate activity
(6.25 mg/mL). Compounds 16 and 17 showed highest activity
equal to that of kanamycin B against P. fluorescens (3.125 mg/
mL) while compounds 5, 7, 9, 12, 13, 15, 19 and 20 exhibited
moderate activity (6.25 mg/mL). Compounds 13 and 17
showed good activity against E. coli (6.25 mg/mL) while com-
pounds 12e16 and 18e20 displayed moderate activity
(12.5 mg/mL). In addition, compound 2 which has the structure
of 1,4-benzodioxin showed better antibacterial activity against
the four tested strains compared to luteolin.
Further SAR studies and mechanistic studies on this new
class of antibacterial compounds are currently under active
investigation and will be reported in due course.
5. Experimental protocols
5.1. Chemistry
Reaction and the resulted products were monitored by thin-
layer chromatography (TLC) on Merck pre-coated silica gel
F254 plates with separated compounds visualized at 254 nm
under a UV lamp. Melting points (uncorrected) were deter-
mined on a XT4 MP apparatus (Taike Corp, Beijing, China).
ESI mass spectra were obtained on a Mariner System 5304
Among compounds prepared, compounds 12 and 13
showed higher antibacterial activity than lots of other com-
pounds. This result disclosed that compounds with aromatic
rings at C-7 position of luteolin were more active than com-
pounds with aliphatic chains. Compounds 15e17 had a slight
difference in the structure, all of them contained a morpholine
1
mass spectrometer, and H NMR spectra were recorded in
DMSO-d6 on a Bruker DPX500 or DPX300 spectrometer

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P.-C. Lv et al. / European Journal of Medicinal Chemistry 44 (2009) 908e914
OH
OH
O
O
O
O
HO
HO
O
O
i
OH
OH
2
1
ii
O
O
O
O
O
O
O
O
O
O
Br
R
iii
OH
OH
4-20
3
13 R=
14 R=
NH
4 R=
N
N
F
N
5 R=
6 R=
N
N
15 R=
16 R=
O
O
NH
NH
7 R=
8 R=
NH
NH
NH
N
17 R=
18 R=
O
OH
9 R=
NH
N
10 R=
NH
NH
N
19 R=
20 R=
HN
11 R=
12 R=
N
N
NH
Scheme 1. Synthesis route of luteolin derivatives. Reagents and conditions: (i) BrCH2CH2Br, K2CO3, DMF, 70 ꢀC, 30 min; (ii) BrCH2CH2Br, K2CO3, DMF,
120 ꢀC, 2 h; (iii) alkylamines and aromatic amines, DMF, 80 ꢀC, 1 h.
with solvent signals allotted as internal standard. Elemental
analyses were performed on a CHNeO-Rapid instrument
and were within ꢁ0.4% of the theoretical values. The reaction
flask was positioned in the maximum-energy area in the
cleaner with cycled water running to control the temperature
of the water bath. The reagents (chemicals), all being of AR
grade, were purchased from Shanghai Chemical Reagent
Company (Shanghai, China). Luteolin (1, >96%, mp 328e
330 ꢀC) provided by Shanxi Huike Botanical Development
Co. Ltd. was used without purification. The antibacterial activ-
ity of reference compounds kanamycin B (Nanjing Zhuyan
Biotechnology Co. Ltd, Amresco 060D0504, Nanjing

P.-C. Lv et al. / European Journal of Medicinal Chemistry 44 (2009) 908e914
911
Table 1
Minimum inhibitory concentrations (MIC e mg/mL) of luteolin derivatives
mixture was distilled to form yellow solid. Recrystallization
of the solid from 20 mL acetone gave compound 3, yellow
powder, 0.282 g, yield 85%, mp: 278e279 ꢀC. 1H NMR
(DMSO-d6): 3.83 (t, J ¼ 5.0 Hz, 2H), 4.32 (dd, J ¼ 5.0 Hz,
8.5 Hz, 4H), 4.45 (t, J ¼ 8.5 Hz, 2H), 6.39 (d, J ¼ 2.0 Hz,
1H), 6.86 (d, J ¼ 2.0 Hz, 1H), 6.93 (s, 1H), 7.03 (d,
J ¼ 8.1 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H).
ESI-MS: 419.1 ([M þ H]þ). Anal. Calcd for C19H15BrO6: C,
54.43%; H, 3.61%. Found: C, 54.53%; H, 3.71%.
Compound
Microorganisms
Gram-positive
B. subtilis
Gram-negative
S. aureus
P. fluorescens
E. coli
1
25
50
25
25
50
50
2
12.5
12.5
12.5
12.5
12.5
12.5
12.5
12.5
6.25
6.25
3.125
1.562
6.25
3.125
1.562
1.562
6.25
6.25
6.25
0.39
1.562
12.5
12.5
12.5
6.25
12.5
6.25
12.5
6.25
12.5
12.5
6.25
6.25
12.5
6.25
3.125
3.125
12.5
6.25
6.25
3.125
6.25
3
25
25
25
25
4
5
12.5
25
25
25
6
5.4. General experimental procedure for the synthesis of
compounds 4e20
7
25
25
25
25
8
9
10
25
25
25
12.5
12.5
6.25
3.125
12.5
6.25
6.25
3.125
12.5
12.5
12.5
1.562
1.562
To a solution of 3 (0.418 g, 1 mmol) in 20 mL of anhydrous
DMF was added different alkylamines and aromatic amines
(10 mmol), followed by heating at 80 ꢀC for 1 h until the start-
ing material disappeared. To the reaction mixture was added
ice water, dropwise. The mixture was filtered, washed with
water, dried over Na2SO4 and concentrated. The residue was
purified with a silica gel column and was eluted with ethyl
acetate:petroleum ether ¼ 1:8 to afford corresponding
products.
11
12
25
12.5
12.5
12.5
12.5
12.5
6.25
12.5
12.5
12.5
3.125
6.25
13
14
15
16
17
18
19
20
Kanamycin B
Penicillin G
5.4.1. Synthesis of 7-(2-(diethylamino)ethoxy)-2-
(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-hydroxy-
4H-chromen-4-one (4)
Yellow powder, yield 80%, mp: 147e149 ꢀC. 1H NMR
(DMSO-d6): 1.06 (t, J ¼ 7.0 Hz, 6H), 3.17e3.20 (m, 4H),
3.44 (t, J ¼ 5.0 Hz, 2H), 4.20 (d, J ¼ 5.0 Hz, 4H), 4.50 (t,
J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.87 (d,
J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.04 (d, J ¼ 8.1 Hz, 1H), 7.63
(d, J ¼ 7.8 Hz, 2H), 12.90 (s, 1H). ESI-MS: 412.1
([M þ H]þ). Anal. Calcd for C23H25NO6: C, 67.14%; H,
6.12%; N, 3.40%. Found: C, 67.28%; H, 6.24%; N, 3.34%.
210002, China) and penicillin G (North China Pharmaceutical
Co. Ltd, D0211107, Hebei 050015, China) were included.
5.2. Experimental procedure for the synthesis of
2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5,7-
dihydroxy-4H-chromen-4-one (2)
To a solution of 1 (0.286 g, 1 mmol) in 20 mL of anhydrous
DMF was added 1,2-dibromoethane (0.38 g, 2 mmol) and po-
tassium carbonate (0.07 g, 0.5 mmol), followed by heating at
70 ꢀC for 30 min. To the reaction mixture was added ice water,
dropwise. The mixture was filtered, washed with water, dried
over Na2SO4 and concentrated. The residue was purified with
a silica gel column and was eluted with ethyl acetate:petro-
leum ether ¼ 1:8 to afford 2, yellow powder, yield 78%, mp:
307e309 ꢀC. 1H NMR (DMSO-d6): 4.32 (d, J ¼ 5.0 Hz,
4H), 6.19 (d, J ¼ 1.8 Hz, 1H), 6.50 (d, J ¼ 2.1 Hz, 1H), 6.85
(s, 1H), 7.01 (d, J ¼ 8.1 Hz, 1H), 7.56 (d, J ¼ 7.8 Hz, 2H),
10.82 (s, 1H), 12.88 (s, 1H). ESI-MS: 313.1 ([M þ H]þ).
Anal. Calcd for C17H12O6: C, 65.39%; H, 3.87%. Found: C,
65.57; H, 3.76%.
5.4.2. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-7-(2-(dipropylamino)ethoxy)-5-
hydroxy-4H-chromen-4-one (5)
Yellow powder, yield 82%, mp: 156e157 ꢀC. 1H NMR
(DMSO-d6): 0.90 (t, J ¼ 7.2 Hz, 6H), 1.42e1.44 (m, 4H),
2.46 (t, J ¼ 7.1 Hz, 4H), 3.43 (t, J ¼ 5.0 Hz, 2H), 4.32 (d,
J ¼ 5.0 Hz, 4H), 4.50 (t, J ¼ 8.5 Hz, 2H), 6.38 (d,
J ¼ 2.0 Hz, 1H), 6.86 (d, J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.05
(d, J ¼ 8.1 Hz, 1H), 7.65 (d, J ¼ 7.8 Hz, 2H), 12.87 (s, 1H).
ESI-MS: 440.2 ([M þ H]þ). Anal. Calcd for C25H29NO6: C,
68.32%; H, 6.65%; N, 3.19%. Found: C, 68.45%; H, 6.58%;
N, 3.24%.
5.3. Experimental procedure for the synthesis of
7-(2-bromoethoxy)-2-(2,3-dihydrobenzo[b][1,4]
dioxin-6-yl)-5-hydroxy-4H-chromen-4-one (3)
5.4.3. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-5-hydroxy-7-(2-(propylamino)ethoxy)-
4H-chromen-4-one (6)
Yellow powder, yield 81%, mp: 160e162 ꢀC. 1H NMR
(DMSO-d6): 0.97 (t, J ¼ 7.2 Hz, 3H), 1.46 (m, 2H), 2.57 (t,
J ¼ 7.1 Hz, 2H), 3.64 (t, J ¼ 5.0 Hz, 2H), 4.13 (d, J ¼ 5.0 Hz,
4H), 4.34 (t, J ¼ 8.5 Hz, 2H), 6.37 (d, J ¼ 2.0 Hz, 1H), 6.83
(d, J ¼ 2.0 Hz, 1H), 6.92 (s, 1H), 7.05 (d, J ¼ 8.1 Hz, 1H),
7.62 (d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H). ESI-MS: 398.1
To a solution of 2 (0.312 g, 1 mmol) in 20 mL of anhydrous
DMF was added 1,2-dibromoethane (4.7 g, 25 mmol) and po-
tassium carbonate (0.14 g, 1 mmol), followed by heating at
120 ꢀC for 2 h until the starting material 2 disappeared. To
the reaction mixture was added ice water, dropwise. The

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P.-C. Lv et al. / European Journal of Medicinal Chemistry 44 (2009) 908e914
([M þ H]þ). Anal. Calcd for C22H23NO6: C, 66.49%; H,
(s, 1H), 3.44 (t, J ¼ 5.0 Hz, 2H), 4.20 (d, J ¼ 5.0 Hz, 4H),
4.50 (t, J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.87 (d,
J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.04 (d, J ¼ 8.1 Hz, 1H), 7.63
(d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H). ESI-MS: 438.1
([M þ H]þ). Anal. Calcd for C25H27NO6: C, 68.63%; H,
6.22%; N, 3.20%. Found: C, 68.75%; H, 6.34%; N, 3.38%.
5.83%; N, 3.52%. Found: C, 66.57%; H, 5.95%; N, 3.47%.
5.4.4. Synthesis of 7-(2-(butylamino)ethoxy)-2-
(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-
hydroxy-4H-chromen-4-one (7)
Yellow powder, yield 83%, mp: 166e167 ꢀC. 1H NMR
(DMSO-d6): 0.90 (t, J ¼ 7.2 Hz, 3H), 1.30e1.34 (m, 2H),
1.52e1.57 (m, 2H), 2.08 (t, J ¼ 7.1 Hz, 2H), 3.66 (t,
J ¼ 5.0 Hz, 2H), 4.16 (d, J ¼ 5.0 Hz, 4H), 4.38 (t, J ¼ 8.5 Hz,
2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.83 (d, J ¼ 2.0 Hz, 1H), 6.92
(s, 1H), 7.02 (d, J ¼ 8.1 Hz, 1H), 7.61 (d, J ¼ 7.8 Hz, 2H),
12.90 (s, 1H). ESI-MS: 412.1 ([M þ H]þ). Anal. Calcd for
C23H25NO6: C, 67.14%; H, 6.12%; N, 3.40%. Found: C,
67.25%; H, 6.24%; N, 3.37%.
5.4.9. Synthesis of 7-(2-(benzylamino)ethoxy)-
2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-
hydroxy-4H-chromen-4-one (12)
Yellow powder, yield 80%, mp: 137e139 ꢀC. 1H NMR
(DMSO-d6): 3.45 (d, J ¼ 5.0 Hz, 2H), 3.83 (t, J ¼ 5.0 Hz,
2H), 4.32 (dd, J ¼ 5.0 Hz, 8.5 Hz, 4H), 4.45 (t, J ¼ 8.5 Hz,
2H), 6.39 (d, J ¼ 2.0 Hz, 1H), 6.86 (d, J ¼ 2.0 Hz, 1H), 6.93
(s, 1H), 7.03 (d, J ¼ 8.1 Hz, 1H), 7.23e7.33 (m, 5H), 7.63
(d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H). ESI-MS: 446.1
([M þ H]þ). Anal. Calcd for C26H23NO6: C, 70.10%; H,
5.20%; N, 3.14%. Found: C, 70.19%; H, 5.41%; N, 3.26%.
5.4.5. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-5-hydroxy-7-(2-(isobutylamino)ethoxy)-
4H-chromen-4-one (8)
Yellow powder, yield 83%, mp: 168e170 ꢀC. 1H NMR
(DMSO-d6): 0.91 (t, J ¼ 7.1 Hz, 6H), 1.62e1.65 (m, 1H), 2.52
(t, J ¼ 7.1 Hz, 2H), 3.44 (t, J ¼ 5.0 Hz, 2H), 4.20 (d, J ¼
5.0 Hz, 4H), 4.50 (t, J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H),
6.87 (d, J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.04 (d, J ¼ 8.1 Hz,
1H), 7.63 (d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H). ESI-MS: 412.1
([M þ H]þ). Anal. Calcd for C23H25NO6: C, 67.14%; H,
6.12%; N, 3.40%. Found: C, 67.28%; H, 6.29%; N, 3.32%.
5.4.10. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-7-(2-(4-fluorobenzylamino)ethoxy)-5-hydroxy-
4H-chromen-4-one (13)
Yellow powder, yield 81%, mp: 139e140 ꢀC. 1H NMR
(DMSO-d6): 3.43 (d, J ¼ 5.0 Hz, 2H), 3.82 (t, J ¼ 5.0 Hz,
2H), 4.29 (dd, J ¼ 5.0 Hz, 8.5 Hz, 4H), 4.46 (t, J ¼ 8.5 Hz,
2H) 6.37 (d, J ¼ 2.0 Hz, 1H), 6.88 (d, J ¼ 2.0 Hz, 1H), 6.96
(s, 1H), 7.03 (d, J ¼ 8.1 Hz, 1H), 7.12 (d, J ¼ 2.0 Hz, 2H),
7.39 (d, J ¼ 8.1 Hz, 2H), 7.64 (d, J ¼ 7.8 Hz, 2H), 12.83 (s,
1H). ESI-MS: 464.1 ([M þ H]þ). Anal. Calcd for
C26H22FNO6: C, 67.38%; H, 4.78%; N, 3.02%. Found: C,
67.45%; H, 4.86%; N, 3.16%.
5.4.6. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-5-hydroxy-7-(2-(2-hydroxyethylamino)ethoxy)-
4H-chromen-4-one (9)
Yellow powder, yield 75%, mp: 180e182 ꢀC. 1H NMR
(DMSO-d6): 2.66 (t, J ¼ 6.5 Hz, 2H); 2.91 (t, J ¼ 6.5 Hz,
2H), 3.43 (t, J ¼ 5.0 Hz, 2H), 4.32 (d, J ¼ 5.0 Hz, 4H), 4.50
(t, J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.86 (d,
J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.05 (d, J ¼ 8.1 Hz, 1H), 7.65
(d, J ¼ 7.8 Hz, 2H), 12.87 (s, 1H). ESI-MS: 400.1
([M þ H]þ). Anal. Calcd for C21H21NO7: C, 63.15%; H,
5.30%; N, 3.51%. Found: C, 63.27%; H, 5.42%; N, 3.62%.
5.4.11. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-5-hydroxy-7-(2-(piperidin-1-yl)ethoxy)-4H-chromen-
4-one (14)
White powder, yield 82%, mp: 145e146 ꢀC. 1H NMR
(DMSO-d6): 1.46e1.48 (m, 2H), 1.54e1.62 (m, 4H), 2.54
(br s, 4H), 3.83 (t, J ¼ 5.0 Hz, 2H), 4.32 (dd, J ¼ 5.0 Hz,
8.5 Hz, 4H), 4.45 (t, J ¼ 8.5 Hz, 2H) 6.39 (d, J ¼ 2.0 Hz,
1H), 6.86 (d, J ¼ 2.0 Hz, 1H), 6.93 (s, 1H), 7.03 (d,
J ¼ 8.1 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H).
ESI-MS: 424.1 ([M þ H]þ). Anal. Calcd for C24H25NO6: C,
68.07%; H, 5.95%; N, 3.31%. Found: C, 68.15%; H, 5.89%;
N, 3.26%.
5.4.7. Synthesis of 7-(2-(cyclopentylamino)ethoxy)-
2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-hydroxy-
4H-chromen-4-one (10)
Yellow powder, yield 84%, mp: 131e132 ꢀC. 1H NMR
(DMSO-d6): 1.46e1.56 (m, 4H), 1.71e1.78 (m, 4H), 2.64
(s, 1H), 3.44 (t, J ¼ 5.0 Hz, 2H), 4.20 (d, J ¼ 5.0 Hz, 4H),
4.50 (t, J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.87 (d,
J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.04 (d, J ¼ 8.1 Hz, 1H), 7.63
(d, J ¼ 7.8 Hz, 2H), 12.90 (s, 1H). ESI-MS: 424.1
([M þ H]þ). Anal. Calcd for C24H25NO6: C, 68.07%; H,
5.95%; N, 3.31%. Found: C, 68.24%; H, 5.78%; N, 3.45%.
5.4.12. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]-
dioxin-6-yl)-5-hydroxy-7-(2-morpholinoethoxy)-
4H-chromen-4-one (15)
Yellow powder, yield 84%, mp: 163e164 ꢀC. 1H NMR
(DMSO-d6): 1.91e2.10 (m, 2H), 2.43e2.61 (m, 6H), 3.76 (t,
J ¼ 5.0 Hz, 2H), 4.29 (dd, J ¼ 5.0 Hz, 8.5 Hz, 4H), 4.49 (t,
J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.84 (d,
J ¼ 2.0 Hz, 1H), 6.93 (s, 1H), 7.03 (d, J ¼ 8.1 Hz, 1H), 7.63
(d, J ¼ 7.8 Hz, 2H), 12.91 (s, 1H). ESI-MS: 426.1
([M þ H]þ). Anal. Calcd for C23H23NO7: C, 64.93%; H,
5.45%; N, 3.29%. Found: C, 64.85%; H, 5.59%; N, 3.26%.
5.4.8. Synthesis of 7-(2-(cyclohexylamino)ethoxy)-
2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-5-hydroxy-
4H-chromen-4-one (11)
Yellow powder, yield 84%, mp: 128e130 ꢀC. 1H NMR
(DMSO-d6): 1.11e1.21 (m, 4H), 1.49e1.60 (m, 6H), 2.56

P.-C. Lv et al. / European Journal of Medicinal Chemistry 44 (2009) 908e914
913
5.4.13. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-5-hydroxy-7-(2-(2-morpholinoethylamino)ethoxy)-
4H-chromen-4-one (16)
(d, J ¼ 2.0 Hz, 1H), 7.01 (s, 1H), 7.14 (d, J ¼ 8.1 Hz, 1H),
7.72 (d, J ¼ 7.8 Hz, 2H), 12.91 (s, 1H). ESI-MS: 425.1
([M þ H]þ). Anal. Calcd for C23H24N2O6: C, 65.08%; H,
5.70%; N, 6.60%. Found: C, 65.12%; H, 5.66%; N, 6.55%.
Yellow powder, yield 84%, mp: 169e170 ꢀC. 1H NMR
(DMSO-d6): 1.98e2.13 (m, 2H), 2.31e2.39 (m, 4H), 2.43e
2.61 (m, 6H), 3.82 (t, J ¼ 5.0 Hz, 2H), 4.31 (dd, J ¼ 5.0 Hz,
8.5 Hz, 4H), 4.45 (t, J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz,
1H), 6.84 (d, J ¼ 2.0 Hz, 1H), 6.93 (s, 1H), 7.03 (d,
J ¼ 8.1 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 2H), 12.78 (s, 1H).
ESI-MS: 469.1 ([M þ H]þ). Anal. Calcd for C25H28N2O7: C,
64.09%; H, 6.02%; N, 5.98%. Found: C, 64.15%; H, 5.99%;
N, 5.85%.
5.5. Antibacterial activity
The antibacterial activity of the synthesized compounds
was tested against B. subtilis, E. coli, P. fluorescens and S. au-
reus using MH medium (Muellere-Hinton medium: casein hy-
drolysate 17.5 g, soluble starch 1.5 g, beef extract 1000 mL).
The MICs of the test compounds were determined by a color-
imetric method using the dye MTT [18]. A stock solution of
the synthesized compound (50 mg/mL) in DMSO was prepared
and graded quantities of the test compounds were incorporated
in specified quantity of sterilized liquid medium (MH medium
for antibacterial activity). A specified quantity of the medium
containing the compound was poured into microtitration
plates. Suspension of the microorganism was prepared to con-
tain approximately 105 cfu/mL and applied to microtitration
plates with serially diluted compounds in DMSO to be tested
and incubated at 37 ꢀC for 24 h. After the MICs were visually
determined on each of the microtitration plates, 50 ml of PBS
(phosphate buffered saline 0.01 mol/L, pH 7.4, Na2H-
PO4$12H2O (2.9 g), KH2PO4 (0.2 g), NaCl (8.0 g), KCl
(0.2 g), distilled water (1000 mL)) containing 2 mg of MTT/
mL was added to each well. Incubation was continued at
room temperature for 4e5 h. The content of each well was re-
moved, and 100 mL of isopropanol containing 5% 1 mol/L
HCl was added to extract the dye. After 12 h of incubation
at room temperature the optical density (OD) was measured
with a microplate reader at 570 nm. The observed MICs are
presented in Table 1.
5.4.14. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-5-hydroxy-7-(2-(3-morpholinopropylamino)ethoxy)-
4H-chromen-4-one (17)
Yellow powder, yield 84%, mp: 174e176 ꢀC. 1H NMR
(DMSO-d6): 1.92e2.10 (m, 4H), 2.33e2.41 (m, 6H), 2.46e
2.58 (m, 6H), 3.84 (t, J ¼ 5.0 Hz, 2H), 4.34 (dd, J ¼ 5.0 Hz,
8.5 Hz, 4H), 4.48 (t, J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz,
1H), 6.84 (d, J ¼ 2.0 Hz, 1H), 6.93 (s, 1H), 7.03 (d,
J ¼ 8.1 Hz, 1H), 7.63 (d, J ¼ 7.8 Hz, 2H), 12.87 (s, 1H).
ESI-MS: 483.1 ([M þ H]þ). Anal. Calcd for C26H20N2O7: C,
64.72%; H, 6.27%; N, 5.81%. Found: C, 64.66%; H, 6.31%;
N, 5.87%.
5.4.15. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-5-hydroxy-7-(2-(pyrrolidin-1-yl)ethoxy)-4H-chromen-
4-one (18)
Yellow powder, yield 77%, mp: 149e150 ꢀC. 1H NMR
(DMSO-d6): 1.62 (br s, 4H), 2.61 (br s, 2H), 2.76 (m, 4H),
3.44 (t, J ¼ 5.0 Hz, 2H), 4.20 (d, J ¼ 5.0 Hz, 4H), 4.50 (t,
J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.87 (d,
J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.04 (d, J ¼ 8.1 Hz, 1H), 7.63
(d, J ¼ 7.8 Hz, 2H), 12.88 (s, 1H). ESI-MS: 410.1
([M þ H]þ). Anal. Calcd for C23H23NO6: C, 67.47%; H,
5.66%; N, 3.42%. Found: C, 67.56%; H, 5.56%; N, 3.36%.
Acknowledgment
The work was co-financed by grants (Projects 30672516 &
30772627) from National Natural Science Foundation of China.
5.4.16. Synthesis of 2-(2,3-dihydrobenzo[b][1,4]dioxin-
6-yl)-5-hydroxy-7-(2-(4-methylpiperazin-1-yl)ethoxy)-
4H-chromen-4-one (19)
References
Yellow powder, yield 79%, mp: 155e156 ꢀC. 1H NMR
(DMSO-d6): 2.28 (s, 3H), 2.36 (br s, 8H), 3.43 (t,
J ¼ 5.0 Hz, 2H), 4.32 (d, J ¼ 5.0 Hz, 4H), 4.50 (t,
J ¼ 8.5 Hz, 2H), 6.38 (d, J ¼ 2.0 Hz, 1H), 6.86 (d,
J ¼ 2.0 Hz, 1H), 6.94 (s, 1H), 7.05 (d, J ¼ 8.1 Hz, 1H), 7.65
(d, J ¼ 7.8 Hz, 2H), 12.87 (s, 1H). ESI-MS: 439.1
([M þ H]þ). Anal. Calcd for C24H26N2O6: C, 65.74%; H,
5.98%; N, 6.39%. Found: C, 65.82%; H, 5.56%; N, 6.25%.
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